Fluorescence-based duplex-quadruplex competition test to screen for telomerase RNA quadruplex ligands

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Guanine-rich sequences in RNA and DNA can form non-canonical structures known as Guanine quadruplexes (G4).
• The RNA component of telomerase (hTERC) contains a G4-prone region at its 5'-end, which may impede crucial P1 helix formation.
• Modulating the RNA duplex-quadruplex equilibrium is a therapeutic strategy to target this interaction.

Purpose of the Study:

• To develop and validate a fluorescence-based assay for identifying small molecules (ligands) that specifically bind to the hTERC G-rich motif.
• To discover ligands that can disrupt the G4 structure and prevent P1 helix formation in hTERC.
• To assess the selectivity of identified ligands for G4-RNA over other quadruplex structures.

Main Methods:

• Screening of 169 diverse molecules using a novel fluorescence-based assay.
• Assay designed to detect inhibition of P1 helix formation by targeting the hTERC G-rich quadruplex.
• Evaluation of ligand selectivity for quadruplexes versus duplexes and for G4-RNA versus other quadruplexes.

Main Results:

• Eleven out of 169 tested molecules were identified as potent inhibitors of P1 duplex formation.
• Several identified compounds demonstrated high selectivity for quadruplex structures over duplex DNA/RNA.
• A subset of these compounds exhibited a preference for G4-RNA structures compared to other G4 types.

Conclusions:

• A robust fluorescence-based assay was established for identifying G4 ligands targeting the hTERC RNA.
• The assay successfully identified potent inhibitors of hTERC P1 helix formation with notable selectivity profiles.
• The developed method is adaptable for high-throughput screening (HTS) against various quadruplex-forming sequences.